1. Field of the Invention
The present invention relates to genetically-altered plants that are hardy with respect to environmental stresses, such as drought and/or freezing, oversized with respect to vegetative and/or sexual structure (as compared to their normal phenotypic counterparts), and capable of growing in media of high salinity. Such plants also display high meristematic activity, and increased in cellular division activity.
2. Background of the Related Art
The prospects for feeding humanity as we enter the new millennium are formidable. Given the every increasing world population, it remains a major goal of agricultural research to improve crop yield. It also is a major goal of horticultural research to develop non-crop plants which are hardier, such as ornamental plants, grasses, shrubs, and other plants found useful or pleasing to man.
Until recently crop and horticultural improvements depended on selective breeding of plants having desirable characteristics. Such selective breeding techniques, however, were often less than desirable as many plants have within them heterogenous genetic complements that do not result in identical desirable traits of their parents.
Advances in molecular biology have allowed mankind to manipulate the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology. has led to the development of plants with increased pest resistance, plants that are capable of expressing pharmaceuticals and other chemicals, and plants that express beneficial traits. Advantageously such plants not only contain a gene of interest, but remain fertile.
One area of interest of late in plant sciences has been the development of plants with improved stress resistance. In general, plants possess and maintain adaptive mechanisms to ensure survival during periods of adverse environmental conditions. Two commons stresses that plants commonly encounter are freezing and drought, both of which are associated with cellular dehydration. It is known that certain plants contain genes turned on by exposure to cold or prolonged periods of dehydration that encode for products that are directly or indirectly responsible for providing greater resistance to drought and/or freeze than many of their counterparts. A number of genes responsive to heat and water stress have now been characterized (See, e.g., U.S. Pat. Nos. 5,837,545, 5,071,962, 4,707,359). These genes are believed by many to produce certain proteins, such as “Water Stress Proteins”, that are postulated to aid the plants survival. For example, certain plants exposed to stress conditions produce a hormone called abcisic acid (ABA) which helps plants close their stromata, thereby reducing the severity of the stress. Unfortunately, ABA is known to inhibit the formation of new leaves, to cause flowers and fruit to drop off, and to lead to a reduction in yield.
Most tropical plants are not believed to have evolved the ability to tolerate prolonged drought and/or freezing. Conversely, many temperate plants are known to have developed at least some ability to tolerate such conditions. The productivity of plant varieties in dry conditions, and after freeze, differ dramatically. For example, tobacco (Nicotiana spp.) produces fresh leaves that are highly sensitive to drought, and cannot be produced commercially in areas with a limited water supply and high degree of evaporation. In contrast to water stress, very little is known about proteins and genes which participate in freezing tolerance. However, it has been hypothesized that a major component of freeze tolerance may involve tolerance to dehydration (See, e.g., Yelenosky, G. C., Guy, L. (1989) Plant Physiol. 89: 444-451).
Another particular area of interest of late has been the development of plants with improved abilities to grow in salinized soil. Salinization of soil occurs when water supplies contain dissolved salt. Upon evaporation of water from such supplies, salts gradually accumulate in the soil. The progressive salinization of irrigated land compromises the future of agriculture in many of the most productive areas of our planet (Serrano, R., et al., Crit. Rev. Plant Sci., 13:121-138 (1994)). For example, arid regions offer optimal photoperiod and temperature conditions for the growth of most crops, but suboptimal rainfall. Artificial irrigation has solved the problem only in the short term as it has been found that soils in such environments frequently are rapidly salinized. To grow in salinized environments, plants must maintain a much lower ratio of Na+/K+ in their cytoplasm than that present in the soil, preventing the growth of a number of plants, including food crops.
Physiological studies suggest that salt exclusion in the root, and/or salt sequestration in the leaf cell vacuoles, are critical determinants for salt tolerance (Kirsch, M., et al., Plant Mol. Biol., 32:543-547 (1996)). Toxic concentrations of sodium chloride (NaCl) build up first in the fully expanded leaves where NaCl is compartmentalized in the vacuoles. Only after their loading capacity is surpassed, do the cytosolic and apoplasmic concentrations reach toxic levels, ultimately leading to loss of turgor, ergo plant death. It has been suggested that hyperacidification of the vacuolar lumen via the V-ATPase provides extra protons required for a Na+/H+ exchange-activity leading to the detoxification of the cytosol (Tsiantis, M. S., et al., Plant J., 9:729-736 (1996)). Salt stress is known to increase both ATP- and pyrophosphate (PPi)-dependent H+ transport in tonoplast vesicles of, for example, sunflower seedling roots. Salt treatments also induce an amiloride-sensitive Na+/H+ exchange activity (Ballesteros, E., et al., Physiologia Plantarum, 99:328-334 (1997)). In the halophyte Mesembryanthemum crystallinum, high NaCl stimulates the activities of both the vacuolar H+-ATPase (V-ATPase) and a vacuolar Na+/H+ antiporter in leaf cells.
Yet another area of agricultural interest is to improve the yield of crop plants and to improve the aesthetic qualities of certain decorative plants. The yield of a plant crop, and the aesthetics of certain decorative plants, may be improved by growing plants that are larger than the wild-type plant in vegetative and/or reproductive structure, as well as improving the growth rate of plants.
A number of compounds have been touted in the prior art as improving the rate of plant growth and biomass production in useful components of the plant. For example., cyclodextrins applied to tissue culture media has been asserted to improve the rate of cell tissue culture growth by mechanisms including increased cell division (See, e.g., U.S. Pat. No. 6,087,176). Certain plant growth hormones, such as auxins (which promote, among other things, root growth), cytokinins, and gibberellic acid (which promotes, among other things, stem growth) when applied to plant tissues are also known to promote increased cellular division. It also is known in the art that certain growth factors may be used to increase plant and/or plant flower size. Unfortunately, isolation and application of such growth hormones and factors is costly and time consuming.
U.S. Pat. No. 5,859,338 discloses that modification of the CLAVATA1 gene of Arabidopsis thaliana causes a loss normal control of cell division in shoot apical meristems and floral meristems. In either case, the loss of control is said to cause an enlargement of the meristem. In flowers, the enlargement is said to lead to an increase in the number of floral organs, including an increase in carpel number, which increases fruit size and seed number. U.S. Pat. No. 5,859,338 provides clavatal nucleic acids and proteins, and modified clavatal nucleic acids and proteins, to result in altered meristem phenotypes.
U.S. Pat. No. 5,750,862 discloses a method for controlling plant cell growth comprising modulating the level and or catalytic activity of a cell cycle control protein in the plant. In particular the patent discloses that by elevating levels of the protein p34cdc2, regeneration into plants of single or groups of cells can be facilitated. Control of regeneration may also be effectuated by control of regulatory elements which indirectly result in modulation of p34cdc2 activity.
A need, therefore, exists for plants having improved stress resistance to drought and/or freeze, possessing larger size attributes than wild-type counterpart varieties, and having increased tolerance to salt in the soil in which they are growing, and which provide for increased biomass of usefull components.